(1) Field of the Invention
The present invention relates to a peptide mimotope of the non-peptide mycotoxin deoxynivalenol. In particular, the peptide mimotope competes with deoxynivalenol for binding to a monoclonal antibody, is antagonistic to the inhibitory effects of deoxynivalenol on in vitro protein synthesis, and does not elicit antibodies in mice that recognize the deoxynivalenol. The present invention also relates to a method that uses the peptide mimotope to determine whether corn, grains or mixed feed is contaminated with fungi that produces deoxynivalenol. The present invention further relates to transgenic plants resistant to deoxynivalenol.
(2) Description of Related Art
Deoxynivalenol (DON) or vomitoxin or dehydronivalenol is 12,13-epoxy-3,7,15-trichothec-9-en-8-one, which is a mycotoxin of the 12,13-epoxy-trichothecenes class of sesquiterpene mycotoxins. It is produced primarily by the fungus Gibberella zeae (Schwein.) Petch (anamorph=Fusarium graminearum Schwabe), which infects corn, small grains and mixed feeds (Hart et al., J. Agric. Food Chem. 31: 657-659 (183); Hart et al., Plant Dis. 66: 1133-1135 (1982); Neish et al., Can. J. Plant Sci. 61: 811-815 (1981)). At the cellular level, the primary toxic effect of DON is inhibition of protein synthesis by binding to the 60S ribosomal subunit, which interferes with peptidyltransferase (Betina, Chem. Biol. Interact. 71: 105-146 (1989); Weber et al., Biochem. 31: 9350-9354 (1992)). DON can cause anorexia and emesis in animals (Scott et al. Proc. natl. Acad. Sci. USA 89: 5398-5402 (1992)). Other toxic effects of DON include skin irritation, hemorrhaging, hematological changes, human lymphocyte blastogenesis impairment, radiomimetic effects, apoptosis and immunotoxicity (Scott et al. ibid.).
DON is primarily found as a contaminant in grains that are infected with the above fungi. It has also been implicated as a chemical warfare agent. Currently, the only means for eliminating DON from human and animal foodstuffs is to detect DON in food and to remove any contaminated foodstuffs from the food supply. Immunoassays offer several advantages compared to other analytical methods for detecting DON in foodstuffs. Following the development of the first monoclonal antibody to DON (Casale et al., J. Agric. Food Chem. 36: 663-668 (1988)), immunological methods, primarily enzyme-linked immunosorbant assay (ELISA), have been widely used for detection of DON (Pestka et al., Food Technol. 49: 120-128 (1995)). An immunoassay for trichothecenes such as DON is disclosed in U.S. Pat. No. 4,879,248 to Chu et al. and kit comprising the immunoassay is disclosed in U.S. Pat. No. 5,118,612 to Chu et al. The immunoassay and kit are either radio immunoassays (RIA) or enzyme-linked immunosorbant assay ELISA based on a competitive control that is DON. These immunological assays have advantages which include high specificity, ease of use, facile sample preparation, and good sensitivity.
The disadvantages of these immunoassays is that they require the user to handle purified DON which poses a toxicity risk to the user. In addition, chemical conjugation of DON to a carrier protein or an enzyme has low efficiency because it involves extensive modification and blocking stages and causes substantial bridge-group interferences and un-wanted cross-reactions (Casale et al., ibid.: Pestka et al., ibid.; Yuan et al., Appl. Environ. Microbiol. 63: 263-269 (1997)). Furthermore, DON is poorly immunogenic and when DON is conjugated to a carrier protein, it""s immunogenicity is only marginally enhanced.
Therefore, it is desirable that an alternative to DON be developed. Preferably, the DON alternative would be non-toxic to the user, not require conjugation to a protein, and be highly immunogenic.
The present invention provides a peptide mimotope of the non-proteinaceous mycotoxin deoxynivalenol (DON). In particular, the peptide mimotope competes with DON for binding to a monoclonal antibody against the DON, is antagonistic to the inhibitory effects of DON on in vitro protein synthesis, and does not elicit antibodies in mice that recognize DON.
The peptide mimotope comprises amino acid sequence SWGPX1PX2 (SEQ ID NO:6) wherein X1 is L, F, or analog thereof and X2 is any amino acid or analog thereof. In particular species of the present invention, a peptide mimotope of DON is provided comprising the amino acid sequence SWGPFPF (SEQ ID NO:2), a peptide mimotope of DON comprising the amino acid sequence SWGPLPF (SEQ ID NO:4), or a peptide mimotope of DON comprising the amino acid sequence SWGPFPFGGGSC (SEQ ID NO:5). The peptide mimotope species are antagonistic to the inhibitory effects of DON on in vitro protein synthesis. In a preferred embodiment, the peptide mimotope is conjugated to a reporter for an immunological assay wherein the reporter is selected from the group consisting of alkaline phosphatase, horseradish peroxidase, or fluorescence molecule. In another preferred embodiment, the peptide mimotope is a part of a peptide or polypeptide. In particular, as a fusion polypeptide wherein the polypeptide is selected from the group consisting of alkaline phosphatase and horseradish peroxidase.
The present invention further provides a nucleic acid that encodes the peptide mimotope of DON comprising an amino acid sequence selected from the group consisting of SWGPLPF (SEQ ID NO:2), SWGPFPF (SEQ ID NO:4), and SWGPFPFGGGSC (SEQ ID NO:5). In particular embodiments, the nucleic acid sequence is selected from the group consisting of SEQ ID NO:1 and SEQ ID NO:3.
The present invention also provides a clone in a microorganism expressing a peptide mimotope of DON comprising amino acid sequence SWGPX1PX2 (SEQ ID NO:6) wherein X1 is L, F, or analog thereof and X2 is any amino acid or analog thereof. In particular species, the peptide mimotope comprises an amino acid sequence selected from the group consisting of SWGPFPF (SEQ ID NO:2), SWGPLPF (SEQ ID NO:4), and SWGPFPFGGGSC (SEQ ID NO:5). In particular, the peptide mimotope expressed by the clone is antagonistic to the inhibitory effects of DON on in vitro protein synthesis. For the clone expressing the peptide mimotope, the peptide mimotope is encoded by a nucleic acid in a plasmid or by a nucleic acid in a recombinant virus vector such as a bacteriophage and the peptide mimotope can be expressed as an isolated peptide or as a part of a fusion polypeptide. Furthermore, the microorganism containing the clone expressing the peptide mimotope can be selected from the group consisting of bacteria and yeasts.
The present invention further provides a transgenic plant containing a nucleic acid that expresses a peptide mimotope of DON that binds to a monoclonal antibody against DON and is antagonistic to the inhibitory effects of DON on in vitro protein synthesis. In particular, the present invention provides a transgenic plant that expresses a peptide mimotope of DON comprising amino acid sequence SWGPX1PX2 wherein X1 is L, F, or analog thereof and X2 is any amino acid or analog thereof. In particular species, the amino acid sequence is selected from the group comprising SWGPFPF (SEQ ID NO:2), SWGPLPF (SEQ ID NO:4), and SWGPFPFGGGSC (SEQ ID NO:5). Further, the peptide mimotope that is expressed can be as an isolated peptide or as a part of a fusion polypeptide.
The present invention also provides an improvement in a method for determining whether a sample contains DON which comprises providing a monoclonal antibody against the DON, reacting the monoclonal antibody with the sample in a reaction mixture containing a labeled DON as a competitor, and determining whether the sample contains DON, wherein the improvement is providing as the competitor a peptide mimotope of DON. In particular, the peptide mimotope has amino acid sequence SWGPX1PX2 (SEQ ID NO:6) wherein X1 is L, F, or analog thereof and X2 is any amino acid or analog thereof. In particular species, the amino acid sequence is selected from the group consisting of SWGPFPF (SEQ ID NO:2), SWGPLPF (SEQ ID NO:4), and SWGPFPFGGGSC (SEQ ID NO:5). Further, the peptide mimotope can be as an isolated peptide or as a part of a fusion polypeptide.
The present invention also provides a method for determining whether a sample contains deoxynivalenol (DON) which comprises: (a) incubating in a reaction the sample, a monoclonal antibody against the DON, and a peptide mimotope which is a competitor of the DON for the monoclonal antibody; (b) detecting in the reaction a complex consisting of the DON bound by the monoclonal antibody and a complex formed by the mimotope and monoclonal antibody; and (c) comparing an amount of each of the complexes wherein a decrease in the amount of the complex comprising the peptide mimotope indicates that the sample contains DON. In particular, the peptide mimotope comprises amino acid sequence SWGPX1PX2 (SEQ ID NO:6) wherein X1 is L, F, or analog thereof and X2 is any amino acid or analog thereof. In particular species, the amino acid sequence is selected from the group consisting of SWGPFPF (SEQ ID NO:2), SWGPLPF (SEQ ID NO:4), and SWGPFPFGGGSC (SEQ ID NO:5). In a preferred embodiment of the method, the monoclonal antibody is produced by hybridoma cell line 6F5. The method further comprises the peptide mimotope which is conjugated to an enzyme selected from the group consisting of horseradish peroxidase and alkaline phosphatase; the peptide mimotope conjugated to a fluorescent reporter; and the peptide mimotope wherein the amino acid of the peptide mimotope is conjugated to an enzyme selected from the group consisting of horseradish peroxidase and alkaline phosphatase to make a fusion protein. Alternatively, the peptide mimotope can be part of a fusion polypeptide wherein the polypeptide is an enzyme that is used as a reporter enzyme in immunoassays, in particular alkaline phosphatase or horseradish peroxidase.
The present invention also provides a kit for determining whether a sample contains deoxynivalenol (DON) comprising: (a) a monoclonal antibody against the DON; (b) a peptide mimotope of the DON that competes with DON for binding to the monoclonal antibody; and (c) instructions for using the kit. In particular, the mimotope comprises amino acid sequence SWGPX1PX2 (SEQ ID NO:6) wherein X1 is L, F, or analog thereof and X2 is any amino acid or analog thereof. In particular species, the peptide mimotope of the kit comprises an amino acid sequence selected from the group consisting of SWGPFPF (SEQ ID NO:2), SWGPLPF (SEQ ID NO:4), and SWGPFPFGGGSC (SEQ ID NO:5). In the preferred embodiment, the monoclonal antibody is produced by hybridoma cell line 6F5. The kit further comprises the peptide mimotope which is conjugated to an enzyme selected from the group consisting of horseradish peroxidase and alkaline phosphatase; the peptide mimotope conjugated to a fluorescent reporter; and a fusion protein wherein the amino acid sequence comprising the peptide mimotope is conjugated to an enzyme selected from the group consisting of horseradish peroxidase and alkaline phosphatase. Alternatively, the peptide mimotope can be part of a fusion polypeptide wherein the polypeptide is an enzyme that is used as a reporter enzyme in immunoassays, in particular alkaline phosphatase or horseradish peroxidase.
The present invention further provides a method for making a plant resistant to deoxynivalenol (DON) comprising introducing into the plant plant""s genome a nucleic acid that encodes a peptide mimotope, which binds to a monoclonal antibody against DON and is antagonistic to the inhibitory effects of DON on in vitro protein synthesis, which is operably linked to a transcription promoter. In particular, the peptide mimotope comprises amino acid sequence SWGPX1PX2 (SEQ ID NO:6) wherein X1 is L, F, or analog thereof and X2 is any amino acid or analog thereof. In a particular species, the peptide mimotope comprises an amino acid sequence selected from the group consisting of SWGPFPF (SEQ ID NO:2), SWGPLPF (SEQ ID NO:4), and SWGPFPFGGGSC (SEQ ID NO:5). The peptide mimotope can be expressed as an isolated peptide or as a part of a fusion polypeptide.
The present invention also provides a method for treating an organism exposed to deoxynivalenol (DON) comprising treating the organism with a peptide mimotope of DON which is antagonistic to the inhibitory effects of DON on in vitro protein synthesis. In particular, the peptide mimotope comprises amino acid sequence SWGPX1PX2 (SEQ ID NO:6) wherein X1 is L, F, or analog thereof and X2 is any amino acid or analog thereof. In a particular species, the peptide mimotope comprises an amino acid sequence selected from the group consisting of SWGPFPF (SEQ ID NO:2), SWGPLPF (SEQ ID NO:4), and SWGPFPFGGGSC (SEQ ID NO:5). The treatment may be given orally, topically, or intravenously. Furthermore, the treatment can comprise a peptide mimotope of DON, which is a vaccine that elicits antibodies against DON. The vaccine can be administered either as a therapeutic treatment to an animal or person displaying symptoms of exposure to DON or as a prophylactic treatment to prevent symptoms caused by a subsequent exposure to DON. The peptide mimotope can be the isolated peptide or as a part of a fusion polypeptide.
It is therefore an object of the present invention to provide a peptide mimotope of deoxynivalenol (DON) for use in immunological assays for detecting DON in a sample.
It is also an object of the present to provide a transgenic plant which is resistant to the affects of DON.
It is a further object of the present invention to provide a method for treating an animal or person exposed to DON.